This study is aimed at understanding the mechanism of action of the p53 tumor suppressor gene in its normal and mutant forms. Inactivation of the endogenous p53 gene is associated with over half of all cases of human cancer. In many tumors, this inactivation occurs through point mutations within the protein coding region, leading to the production of full length mutant p53. At least some of those mutant p53 forms may actually gain a novel function, which contributes directly to cancer development. The long term goal of this project is to understand the molecular basis for the diverse biological activities of p53, and to elucidate the signalling pathways which regulate the activity of the p53 protein in normal and malignant cells. Given the frequent association between p53 alerations and human cancer, such knowledge may eventually provide valuable for devising new diagnostic and therapeutic strategies. One aspect of the work is based on the fact that p53 is a sequence specific transcriptional activator, capable of triggering selectively the expression of particular target genes. An attempt will therefore be made to gain insight into the model of action of p53 through cloning, identifying and studying in detail several p53 target genes. Through functional assays, based either on the overexpression of each target genes or on its down-regulation through antisense RNA, the potential contributions of these target genes to the effects of p53 will be evaluated. An additional effort will center on the possibility that the Mdm2 protein, itself the product of a p53 target gene, is involved in the regulation of p53 stability in the cell. Mdm2 expression levels will be manipulated up or down, through sense and anti-sense RNA, respectively. Use will also be made of mdm2 "knock-out" cells. In each case, the question will be asked whether the stability of endogenous p53 is affected by the levels of cellular Mm2. An attempt will also be made to find out whether the effect of dm2 on p53 stability can account for a variety of older observations regarding changes in p53 turn-over rates under various conditions. In a third project, the potential gain of function oncogenic effect of mutant p53 will be investigated. In particular, it will be asked whether such p53 mutants can promote cancer by increasing the cell's resistance to apoptosis-inducing signals, including exposure to DNA damaging agents in use for cancer therapy. Finally, new transgenic mouse strains will be generated and explored toward learning how the biochemical activity of p53 is regulated within an intact animal. Such regulation will be studied during development, as well as in cancer progression.